This research is directed toward the goal of understanding the structural organization of genomic DNA and associated nuclear proteins through detailed three-dimensional analysis of structures such as the nucleosome core particle, a small gene complete with promoter sequence, and more complex structures like the chromatosome and the polynucleosomal 30 nanometer fiber. The primary objective is to determine a medium resolution crystallographic structure of reconstituted nucleosome core particles containing a defined- sequence DNA. Nucleosomes reconstituted from purified chicken erythrocyte histones and a defined-sequence DNA have been crystallized. The structure will be solved to a resolution of approximately 3 Angstroms using data collected at a synchrotron light source. A combination of heavy-atom derivative multiple-wavelength anomalous diffraction and molecular replacement will be used to phase the X-ray diffraction data. The defined-sequence DNA of choice, constructed from "one-half" of a 145 base-pair human alpha-satellite sequence, is a 146 base pair palindrome which confers overall two-fold symmetry to the nucleosome core particle. The palindrome-containing, two-fold symmetric nucleosomes display the highest resolution diffraction of any nucleosome crystals. Another defined-sequence DNA consists of an entire tRNA-Lys gene with the polymerase III promoter site located near the center of the nucleosome as in vivo. The entire gene and its internal promoter region will be observed in this structure. Other defined-sequence DNAs which form precisely phased nucleosomes for potential structure determination are a human alpha- satellite repeat and a sequence from SV40 flanking the nucleosome free region. This research program will eventually provide the near-atomic resolution information which is of fundamental importance to understanding the structures of DNA and chromatin as well as the controlling role of nucleosomes in modulating gene expression, retroviral integration target site selection, and DNA replication. The structural information attained will contribute to an understanding of the molecular basis of carcinogenesis, and should also be of interest to the Human Genome Project.